1␣,25(OH) 2 D 3 is an important negative regulator of parathyroid hormone (PTH) gene transcription. In parathyroid cells, as in other target tissues, 1␣,25(OH) 2 D 3 is degraded by side chain oxidation by the inducible 24-hydroxylase. We have previously shown that one metabolite of this pathway, 1␣,23(S),25-(OH) 3 -24-oxo-D 3 , potently suppresses PTH synthesis and secretion in cultured bovine parathyroid cells (bPTC). Further examination of the metabolites of 1␣,25(OH) 2 D 3 in bPTC has revealed another compound that is less polar than 1␣,25(OH) 2 D 3 . By HPLC analysis and mass spectrometry, this metabolite was identified as 1␣,25(OH) 2 -3-epi-D 3 . The activity of this metabolite on PTH gene transcription was assessed by the steady-state PTH secretion by bPTC after 72-h treatment with concentrations from 10 Ϫ11 M to 10 Ϫ7 M. 1␣,25(OH) 2 -3-epi-D 3 was found to be only slightly, but not significantly, less active than the native 1␣,25(OH) 2 D 3 in suppressing PTH secretion despite having 30 times lower affinity for the bPTC VDR. Both 1␣,25(OH) 2 D 3 and 1␣,25(OH) 2 -3-epi-D 3 maximally suppressed PTH secretion by 50%. Along with 1␣,25(OH) 2 -3-epi-D 3 , the activities of the other two A-ring diastereomers were assessed. 1,25(OH) 2 D 3 suppressed PTH only at 10 Ϫ7 M with a decrease of only 30%, in good agreement with its low VDR affinity. Surprisingly, 1,25(OH) 2 -3-epi-D 3 stimulated PTH secretion by 30-50% at concentrations from 10 Ϫ11 M to 10 Ϫ 8 M and fell to control (untreated) rates at 10 Ϫ7 M. The mechanism for this increase in PTH secretion is under investigation. Metabolism studies performed in bPTC cells using high concentrations of 1␣,25(OH) 2 D 3 substrate showed that in some incubations, the concentration of 1␣,25(OH) 2 -3epi-D 3 was even higher than that of the parent substrate 1␣,25(OH) 2 D 3 . This finding indicates a slower rate of metabolism for this diastereomer. Thus, production and accumulation of 1␣,25(OH) 2 -3-epi-D 3 , as a major stable metabolite of 1␣,25(OH) 2 D 3 in parathyroid glands, may contribute to the prolonged suppressive effect of 1␣,25(OH) 2 D 3 on PTH gene transcription.